Stabilizers, polymers, and emulsions useful for molecular imprinting technology

ABSTRACT

The claimed invention is directed to stabilizers, polymers, and emulsifiers useful in preparing molecularly imprinted materials having recognition capabilities corresponding to the imprint molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/451,711 filed May26, 1995 now abandoned. The entire contents of this prior applicationare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to molecularly imprinted polymer supportsand a method of making these supports. More specifically the presentinvention involves arranging polymerizable functional monomers around aprint molecule using suspension polymerization techniques which isaccomplished with a stabilizing copolymer having the following formula:##STR1## wherein: X is C_(n) F_(2n+1), C_(n) F_(2n+1) (CH₂)_(r) O--,C_(n) F_(2n+1) O-- or C_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ --O;

Y is (Z)_(t) CH₃ O(CH₂ CH₂ O)_(m) C₂ H₄ --O or (Z)_(t) CH₃ O(CH₂ CH₂O)_(m) ;

Z is a print molecule;

n is between 1 and 20;

m can be zero to about 500;

p is at least 1;

q can be zero or any positive number;

r is 1-20, preferably 1 or 2;

s is zero or 1; and

t is zero or 1.

The polymer support of the present invention is preferably in bead formand is capable of separating or resolving amino acids, amino-acidderivatives, pharmaceutical compounds and poly-saccharides.

BACKGROUND OF THE INVENTION

Molecular imprinting, also referred to as templating, has been used forchiral separations and involves arranging polymerizable functionalmonomers around a print molecule. This is achieved either by utilizingnon-covalent interactions such as hydrogen bonds, ion-pair interactions,etc. (non-covalent imprinting), or by reversible covalent inter-actions(covalent imprinting) between the print molecule and the functionalmonomers. The resulting complexes are then incorporated bypolymerization into a highly cross-linked macroporous polymer matrix.Extraction of the print molecule leaves sites in the polymer withspecific shape and functional groups complementary to the original printmolecule. Mosbach, K., Trends in Biochemical Sciences, Vol. 7, pp.92-96, 1994; Wulff, G., Trends in Biotechnology, Vol. 11, pp. 85-87,1993; and Andersson, et al., Molecular Interactions in Bioseparations(Ngo. T. T. ed.), pp. 383-394.

Different racemic compounds have been resolved via molecular imprinting,i.e., "amino acid derivatives", see Andersson, et al., MolecularInteractions in Bioseparations (Ngo T. T. ed.), Plenum Press, pp.383-394, 1993; "drugs", Fischer, et al., J. Am. Chem. Soc., 113, pp.9358-9360, 1991; Kempe, et al., J. Chromatogr., Vol. 664, pp. 276-279,1994; and "sugars", Wulff, et al., J. Org. Chem., Vol. 56, pp. 395-400,1991; Mayes, et al., Anal. Biochem., Vol. 222, pp. 483-488, 1994.Baseline resolution has been achieved in many cases.

An advantage of molecularly imprinted polymers, in contrast to otherchiral stationary phases, is the predictable order of elution ofenantiomers. Imprintable supports have been prepared from bulkpolymerization techniques, using a porogenic solvent to create a blockof macroporous polymer. However, bulk polymerization supports must becrushed, ground and sieved to produce appropriate particle sizes for usein separatory columns and analytical protocols. For example, inchromatographic evaluations, polymer particles smaller than 25 μm aregenerally used. However, from the bulk polymerization process thegrinding process used to provide these smaller particles from the bulkpolymerization process is unsatisfactory. Grinding produces irregularlyshaped particles and an excessive and undesirable quantities of "fines."Typically less than 50 percent (50%) of the ground polymer is recoveredas useable particles. Irregular particles generally give less efficientcolumn packing for chromatography and often prove troublesome in processscale-up. Hence, uniformly shaped particles, e.g. beaded polymers, wouldbe preferable in most cases. The grinding process also requires anadditional treating step to remove the fines, i.e., sedimentation. Thisis costly and time consuming. The bulk polymerization and necessarygrinding process makes this prior art technique labor intensive,wasteful and unacceptable.

Attempts have been made to use suspension and dis-persion polymerizationtechniques for producing beads from acrylic monomers which can containimprinted molecules. In principle these suspension and dispersionpolymerization techniques should offer an alternative to bulkpolymerization. However, existing suspension and dispersion techniquesare not satisfactory because water or a highly polar organic solvent(e.g. an alcohol) is used as the continuous phase for the relativelyhydrophobic monomers. These solvents are incompatible with most covalentand non-covalent imprinting mixtures due to the competition betweensolvent and functional monomers for specific interaction with the printmolecule. Since suspension polymerization techniques use the solvent inlarge molar excess, the solvents saturate the monomer phase anddrastically reduce the number and strength of the inter-actions betweenfunctional monomers and print molecules. In addition, because of thehigh solubility of acidic monomers in water, random copolymerization ofmonomers and cross-linker is probably not achieved. Water soluble printmolecules are also lost due to partitioning into the aqueous phase. Notunexpectedly, attempts to make molecularly imprinted polymer beads bysuspension polymerization in water have led to only very poorrecognition. Damen, et al., J. Am. Chem. Soc., Vol. 102, pp. 3265-3267,1980; Braun, et al., Chemiker-Zeitung, Vol. 108, pp. 255-257, 1984;Bystrom, et al., J. Am. Chem. Soc., Vol. 115, pp. 2081-2083, 1993. Withstable covalent or metal chelate bonds between functional monomers andprint molecules prior to polymerization, it may be possible to useaqueous conditions.

Attempts have also been made to produce composite beaded particles byimprinting in the pore network of performed beaded silica, Norrlow, etal., J. Chromatogr., Vol. 299, pp. 29-41, 1924; Wulff, et al., ReactivePolymers, Vol. 3, pp. 261-2757, 1985 or TRIM. However, the preparationrequires careful handling and the volume of imprinted polymer per unitcolumn is inevitably reduced by the beads themselves.

Sellergren, B., J. Chromatogr., Vol. 673, pp. 133-141, 1994 andSellergren, B., Anal. Chem., Vol. 66, pp. 1578-1582, 1994, report theuse of dispersion polymerization in a polar solvent mixture formolecular imprinting. The process produces random precipitates ratherthan regular beads. Acceptable results were only achieved for highlycharged print molecules, presumably due to the presence of competingsolvent effects.

Thus, a need exists for a method that produces beaded polymerscontaining molecular imprints that is simple and reproducible, does notcompromise the quality of the imprints obtained and eliminates the needfor grinding and sieving equipment. A need also exists for a molecularimprinted polymer bead that is uniform.

SUMMARY OF THE INVENTION

The present invention relates to molecular imprinted polymer supportsand their preparation via suspension polymerization. The suspensiontechniques according to the present invention provide for molecularimprinting by using a perfluorocarbon liquid containing polyoxyethyleneester groups as the dispersing phase. Theperfluoro-carbon-polyoxyethylene ester containing group compound doesnot interfere with the interactions between functional monomers andprint molecules that are required for the recognition process duringmolecular imprinting. Controllable "support" particle sizes from about 2μm to about 100 μm are obtained by varying the amount of stabilizingpolymer, or agitating technique.

Accordingly, it is an object of the present invention to provide amethod that enables imprinted polymers to be easily produced, in beadedform with an almost quantitative yield of useable material.

It is another object of the present invention to provide a fluorocarboncopolymer that stabilizes the emulsion in suspension polymerizationprocessing without interfering with the interactions between functionalmonomers and print molecules.

It is a still further object to stabilize an emulsion of functionalmonomers, cross-linkers, print molecules, initiators and porogenicsolvents.

Another object of the present invention is to provide molecularlyimprinted polymer bead having a size of about 2 to about 100 μm, in highyield.

A still further object of the present invention is to provide small,about 2-5 μm, beaded packings that provide low back pressure, rapiddiffusion, and good separation at high flow rates.

These and other objects and advantages will become more apparent in viewof the following description and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of bead diameter versus PFPS quantity added for a"standard polymerization" containing 1.84 g EDMA, 0.16 g MAA, 4.2 gchloroform and 20 mg AIBN emulsified in 20 ml PMC.

FIGS. 2a-2e show scanning electronmicrographs of beads produced fromsuspension polymerization in PMC in accordance with the presentinvention where the beads were placed on aluminum pegs and sputtercoated with 15 mm gold using a polaron E5150 coater. The images wereobtained using an ISI 100A SEM at 25 kV. The magnification is 500×. 2(a)PF2; 2(b) PF13; 2(c) PF13; 2(d) PF14; and 2(e) PF15.

FIGS. 3a-3e show HPLC traces showing separation of Boc-D,L-Phe by a 25cm column of Example PF15 (5 μm TRIM beads). Conditions: the column wasequilibrated with chloroform+0.25% acetic acid; 1 mg Boc-D,L-Phe in 20μl mobile phase was injected and eluted with the same solvent at flowrates of 3(a) 0.5 mlmin⁻¹ ; 3(b) 1 mlmin⁻¹ ; 3(c) 2 mlmin⁻¹ ; 3(d) 3mlmin⁻¹ ; and 3(e) 5 ml-min⁻¹. At 0.5 ml-min⁻¹, f/g=0.89, Rs=1.36 andα=1.52 f/g values were 0.89, 0.89, 0.85, 0.76 and 0.61 at 0.5, 1, 2, 3and 5 mlmin⁻¹ respectively.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, a suspension polymerizationtechnique based on emulsion of noncovalent imprinting mixtures formed inliquid perfluorocarbons that contain polyoxyethylene ester groups, isprovided. Most prior art suspension and dispersion techniques use wateror a highly polar organic solvent (e.g. an alcohol) as the continuousphase for the relatively hydrophobic monomers, which is not fullysatisfactory. Accordingly, a different approach is required. The presentinvention provides the "different approach." In addition, the drawbacksof prior art processes, e.g., solvents that are incompatible with mostcovalent and non-covalent imprinting mixtures due to the competitionbetween solvent and functional monomers for specific interaction withthe print molecule are avoided. The present invention avoids the use ofdispersants which interfere with the interactions that are required forrecognition between print molecules and functional monomers. In order tocreate reasonably stable emulsion droplets containing monomers,cross-linkers, print molecules, porogenic solvents, and fluorinatedsurfactants, the invention uses a perfluorocarbon polymer that alsocontains polyoxyethylene ester groups, with or without a print moleculefor providing surface imprinting.

The stabilizing/dispersing agent of the present invention is generallydefined by the following formula: ##STR2## wherein: X is C_(n) F_(2n+1),C_(n) F_(2n+1) (CH₂)_(r) O--, C_(n) F_(2n+1) O-- or C_(n) F_(2n+1) SO₂N(C₂ H₅)C₂ H₄ --O,

Y is (Z)_(t) CH₃ O(CH₂ CH₂ O)_(m) C₂ H₄ --O or (Z)_(t) CH₃ O(CH₂ CH₂O)_(m) ;

Z is a print molecule;

n is between 1 and 20;

m can be zero to about 500;

p is at least 1;

q can be zero or any positive number;

r is 1-20, preferably 1 or 2;

s is zero or 1; and

t is zero or 1.

Block, triblock and multiblock copolymers formed from the acrylatedmonomers of C_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ --OH and (Z)_(t) CH₃ O(CH₂CH₂ O)_(m) C₂ H₄ --OH are also within the scope of the presentinvention. A preferred stabilizing/dispersing agent according to thepresent invention is a copolymer containing monomer A defined by theformula: C_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ O--CO--CH═CH₂ ; and comonomerB defined by the formula: CH₃ O(CH₂ CH₂ O)_(m) C₂ H₄ O--CO--CH═CH₂ ;where n is between 1 and 20 and m is between 0 and 500. Preferably, n isabout 7.5 and m is about 43. Most preferably, the stabilizing/dispersingagent is defined by the following formula, ##STR3## wherein: X is C_(n)F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ --O;

Y is (Z)_(t) CH₃ O(CH₂ CH₂ O)_(m) C₂ H₄ --O;

Z is a print molecule;

n is between 1 and 20;

m can be zero to about 500;

p is at least 1;

q can be zero or any positive number;

s is zero or 1;

t is zero or 1, and

enables the production of spherical beads in high yields via asuspension polymerization process.

Preferably, m=about 7.5, n=about 43, p=about 19 and q=about 1. Thevalues m, n, p, and q need not be whole numbers.

As a result of using the stabilizer of the present invention, thephysical characterization of beads is achieved either by utilizingnon-covalent interactions such as hydrogen bonds, ion-pair interactions,etc. (non-covalent imprinting), or by reversible covalent inter-actions(covalent imprinting) between the print molecule and the functionalmonomers. The complexes formed are then incorporated by polymerizationinto a highly cross-linked macroporous polymer matrix formed from thecopolymerization of different acrylic monomers. Extraction of the printmolecule leaves sites in the polymer with specific shape and functionalgroups complementary to the original print molecule.

The print molecules that can be used in the present invention include,but are not limited to:

1. D- and L-Boc tryptophans

2. D- and L-Boc phenylanalines

3. D- and L-phenylanalines

4. D- and L-Boc-proline-N-hydroxsuccinimide esters

5. D- and L-Cbz tryptophans

6. D- and L-Cbz-aspartic acids

7. D- and L-Cbz-glutamic acids

8. D- and L-Cbz-tryptophan methyl esters

9. nitrophenyl α and β, D-, L-galactosides

10. (S)-(-) timolol

11. D-fructose

12. D-galactose

13. phenyl α-D-mannopyranoside

14. acryl α and β glucosides

15. (R)-phenylsuccinic acid

16. Ac-L-Trp-OEz

17. L-PheβNA

18. L-LeuAn

19. L-PheAn

20. L-PheGlyAn

21. L-MenPheAn

22. L-PyMePheAn

23. L-PLPheAn

24. N-Ac-L-Phe-L-Trp-OMe

25. diazepham

26. propranolol

27. ephidrine

However the Boc- D- and L-Phe, and L-Phe have been used in the followingnon-limiting examples.

REAGENT PREPARATION Monomers

Ethylene glycol dimethacrylate (EDMA) and methacrylic acid (MAA) (Merck,Darmstadt, Germany) were distilled under reduced pressure prior to use.Trimethylolpropane trimethacrylate (TRIM) (Aldrich Chemie, Steinheim,Germany), styrene (Aldrich), methyl methacrylate (MMA) (Aldrich) andbenzyl methacrylate BMA) (Polysciences, Warrington, Mass.) were used asreceived. 2,2'-azobis (2-methylpropionitrile (AIBN) came from JanssenChimica, Goel, Belgium.

2-(N-ethylperfluoroalkylsulphonamido)ethanol (PFA-1) (Fluorochem, OldGlossop, UK), PEG2000 monomethylether (MME) (Fluka Chemic A.G., Buchs,Switzerland) and PEG350MME (Sigma, St. Louis, Mo.) were converted totheir acrylates by reaction with acryloyl chloride and tri-ethylamine indichloromethane, were also available commercially (from Fluorochem andPolysciences respectively). Perfluoro(methylcyclohexane) (PMC) andFluorad FC430 were also obtained from Fluorochem.

Imprint Molecules

Boc-D-Phe, BocU-Phe and Boc-D, L-Phe were obtained from Bachem A.G.,Bubendorf, Switzerland). (Boc(tert-butoxy carbonyl; Phe=phenylanaline).

Porogenic Solvents

Chloroform (CHCl₃) (HPLC grade) was passed down a basic alumina columnto remove ethanol and stored over molecular sieves for use as porogenicsolvent during imprinting. For HPLC it was used as received. Toluene wasdried with sodium and acetonic with molecular sieves prior to use. Othersolvents were of analytical grade or better and were used as received.

PREPARATION OF STABILIZERS

The most emulsion stabilizing/dispersing polymers (PFPS) according tothe present invention are defined by the formula ##STR4## wherein: X isC_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ --O;

Y is (Z)_(t) CH₃ O(CH₂ CH₂ O)_(m) C₂ H₄ --O;

Z is a print molecule;

n is between 1 and 20;

m can be zero to about 500;

p is at least 1;

q can be zero or any positive number; and

t is zero or 1; and

in particular where n is about 7.5, m is about 43, p is about 19 and qis about 1.

EXAMPLE 1

4 g acryloyl PFA-1 (7.2 mmole) C_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄--O--CO--CH═CH₂ and 0.76 g acryloyl PEG2000MME (0.36 mmole) defined bythe following formula: CH₃ O(CH₂ CH₂ O)_(m) C₂ H₄ --O--CO--CH═CH₂, weredissolved in 10 ml of chloroform, where n is about 7.5 and m is about43.24 mg (76 μmole) of AIBN was added and dissolved oxygen removed bynitrogen sparring for 5 minutes. The tube was then sealed and thecontents poly-merized at 60° C. for 48 hours in a shaking water bath.The resulting solution was slightly turbid and became much more turbidon cooling. Most of the solvent was removed by slow evaporation at 30°C. under reduced pressure (to avoid foaming) and the remainder underreduced pressure at 60° C. The resulting polymer was a sticky paleyellow paste which was used without further treatment.

Other polymer stabilizers were synthesized in a similar fashion usingthe appropriate ratio of monomers and 1 mole % of AIBN. All formed creamto amber polymers varying from glassy to very soft pastes depending onthe composition.

Suspension Polymerizations

The amount of porogenic solvent required to just saturate 20 ml PMC wasdetermined. The required amount of PFPS was dissolved in this volume ofporogenic solvent in a 50 ml borosilicate glass tube and 20 ml PMC addedand shaken to give a uniform white to opalescent emulsion. 5 ml of"imprinting mixture", (Table 1) was added and emulsified by stirring at2000 rpm for 5 minutes.

                  TABLE 1                                                         ______________________________________                                                            MAA    EDM            PFPS                                Ex.*  Print Molecule (mg)                                                                         (g)    A(g)  Solvent (g)                                                                            (mg)                                ______________________________________                                        PF1   Boc--L--Phe (120)                                                                           0.16   1.84  CHC1.sub.3 (4.2)                                                                       10                                  PF2   Boc--L--Phe (120)                                                                           0.16   1.84  CHC1.sub.3 (4.2)                                                                       25                                  PF3   Boc--L--Phe (120)                                                                           0.16   1.84  CHC1.sub.3 (4.2)                                                                       50                                  PF4   Boc--L--Phe (120)                                                                           0.16   1.84  CHC1.sub.3 (4.2)                                                                       75                                  PF5   Boc--L--Phe (120)                                                                           0.16   1.84  CHC1.sub.3 (4.2)                                                                       100                                 PF6   Boc--L--Phe (120)                                                                           0.16   1.84  CHC1.sub.3 (4.2)                                                                       200                                 PF7   Boc--L--Phe (120)                                                                           0.16   1.84  CHC1.sub.3 (4.2)                                                                       500                                 PF8   Boc--L--Phe (120)                                                                           0.32   1.84  CHC1.sub.3 (4.2)                                                                       25                                  PF9   Boc--L--Phe (120)                                                                           0.48   1.84  CHC1.sub.3 (4.2)                                                                       25                                  PF10  Boc--L--Phe (120)                                                                           0.64   1.84  CHC1.sub.3 (4.2)                                                                       25                                  PF11  Boc--L--Phe (68)                                                                            0.265  1.84  CHC1.sub.3 (4.2)                                                                       25                                  PF12  Boc--D,L--Phe (120)                                                                         0.16   1.84  CHC1.sub.3 (4.2)                                                                       25                                  PF13  None          0.16   1.84  toluene (2.45)                                                                         25                                  PF14  None          0.16   1.84  acetone (2.25)                                                                         25                                  PF15  Boc--L--Phe (308)                                                                           0.4    1.57  CHC1.sub.3 (4.6)                                                                       100                                                            TRIM                                               PF16  Boc--L--Phe (308)                                                                           0.4    1.57  CHC1.sub.3 (4.6)                                                                       25                                                             TRIM                                               ______________________________________                                         *The beads prepared according to the present invention, as well as the        comparative examples, are designed with the PF code numbers in column 1 o     the table.                                                               

The polymerization apparatus used for all polymerizations in the presentinvention comprised a 50 ml boro-silicate glass tube with screw lid, thecenter of which was drilled to allow the shaft of a stainless steel flatblade stirrer to pass there through. The stirrer blade was aboutone-half the length of the tube. A rubber seal was used to reduceevaporation through this hole. An additional small hole in the lidallowed a nitrogen stream to be fed into the tube via a syringe needle.The tube was held vertically in a retort stand and stirred with anoverhead stirrer. A UV lamp was placed about 5 cm away from the tube andthe lamp and tube surrounded with aluminum foil to maximize reflectedlight.

Emulsions designated PF1-PF16 in Table 1 above were prepared and placedin the reactor which was purged with nitrogen for 5 minutes and then theemulsions polymerized by UV irradiation at 366 nm at room temperatureunder a gentle nitrogen stream stirring at 500 rpm. Polymerization wascontinued for 3 hours. The resulting polymer particles (beads) werefiltered on a sintered glass funnel and the PMC recovered. The beadswere washed extensively with acetone, sonicating to break up looseaggregates of beads (large aggregates were broken up by gentle crushingwith a spatula), before drying and storing.

Varying the amount of PFPS used during the polymerization controls beadsize, the relationship being shown in FIG. 1. FIG. 1 is a graph of themean and standard deviation for beads made in accordance with thepresent invention using different amounts of PFPS. A standard wasprepared with 2 g of monomers in a 5 ml total volume (see Table 1). 10mg PFPS is at the lower limit of the range where stable emulsions can beformed and quite a lot of aggregate was also present in this sample.Using 150 mg or more of PFPS gave only very small irregular particles of1-4 μm. No beads were apparent in these samples.

COMPARATIVE EXAMPLES

Two commercial fluorinated surfactants (polyfluoro-alcohol (PFA-1) andfluorad FC430), a homopolymer of acryloyl PFA-1, a range of randomcopolymers of acryloyl PFA-1 with styrene, methyl methacrylate or benzylmeth-arcylate, and graft copolymers containing perfluoroester groups andPEG groups attached to the main acrylate chain, were also evaluated asstabilizers. Most of these, however, proved to be ineffective. Theprocess was hindered by the high density of the dispersant which causedrapid "creaming" of the emulsion, thus favoring coalescence of disperseddroplets.

Polyacryloyl PFA-1, alone, was evaluated and gave sufficiently stableemulsions for suspension polymerization and gave good quality beads.Unfortunately, due to its poor solubility, this surfactant proved to beextremely difficult to remove from the surface of the beads afterpolymerization, resulting in extremely hydrophobic surfaces.

The most effective emulsion was achieved using a copolymer ofacryloyl-PFA-1 and acryloyl-PEG2000MME (mole ratio 20:1-termed PFPS).

MOLECULARLY IMPRINTED BEAD PROPERTIES Size

The type of emulsification impacts the polymerization process and theresulting molecularly imprinted beads. The preferred method involvedstirring at about 2000 rpms for about 5 minutes and gave good uniformityand reproducibility for a bead having a size of between 2 and 25 μm.Five separate polymerizations performed on different days using 25 mgPFPS as the emulsifier gave a mean bead size of 19.7 μm and a standarderror of 0.6 μm.

Emulsification in an ultrasonic bath for 5 minutes gave a much broadersize distribution with an excessive quantity of small particles.Conventional shaking in a tube 3 or 4 times gave good results if largerbeads were desired, i.e., 40 μm to about 100 μm.

The polymerization temperatures also affected the polymerization processand resulting beads. Attempts to use thermal initiation at 45° C. usingABDV as initiator gave only small irregular fragments. UV initiation ofpolymerization at 4° C. led to a large amount of aggregation. Mostpolymerizations were performed at ambient temperature (about 20° C.),i.e. at least about 18° C., although some temperature increase occurredduring polymerization due to the proximity of the UV lamp.Polymerizations carried out during very warm weather, when ambienttemperature reached 30° C., gave slightly smaller beads, indicating thatpolymerization temperature can also impact bead size while stillproviding reproducible results.

Polymerizations were also carried out in a range of solvents commonlyused in molecular imprinting, e.g., chloroform, toluene, acetonitrileand acetone. The polymerization method can use all of these solvents andhence should be appropriate for most imprinting situations. However, thepreferred solvents are chloroform, toluene and acetone. The size andrace structure of the beads produced also depends on the porogenicsolvent used. Both toluene (26 μm±12 μm-means±SD) and acetone (52 μm±15μm) gave larger beads than chloroform (18 pm±8 pm) for 25 mg of PFPS ina "standard" polymerization.

Dead Size Distributions

Suspensions of beads in acetone were dried onto microscope slides andabout 150 beads measured at random using a calibrated graticule in anoptical microscope. Either 100× or 400× magnifications was useddepending on the particle size. Some samples were also imaged by SEM.Measurements made from these images compared well with the results fromoptical determinations.

Scanning Electron Micrographs

Polymer beads were placed on aluminum pegs and sputter coated with 15 nmgold using a Polaron E5150 gold coater. Images were then obtained usingan ISI 100A SEM at 25 kV in order to compare the sizes, surfaces andpore structures of beads produced under different conditions.

Scanning electron micrographs of some of the beaded polymer preparationsare shown in FIGS. 2a-2e. The method according to the present inventionproduces substantially spherical beads, both for EDMA(2a-2d) andTRIM(2e) based polymers, and using a variety of porogenic solvents. Theincidence of defects, such as surface indentations or small holes, issomewhat higher than is usually observed for water-based suspensionpolymerizations in water. The morphology of the beads is typical ofbeads made by suspension polymerization with a slightly denser andsmoother surface layer covering a more porous structure in the interior.

The beads made using acetone as porogenic solvent (FIG. 2d) differedfrom the others. They were larger, had much rougher surface morphologyand more "debris" on their surfaces than those prepared using chloroformor toluene. The beads made with toluene as porogenic solvent had a lessdense surface shell and somewhat more porous interior structure thanthose made with chloroform. FIG. 2b shows beads of polymer PF9 which hasa lower proportion of cross-linker than that in FIG. 2a (polymer PF2).The beads in FIG. 2b are much more irregular and distorted, suggestingthat these particles might remain softer and deformable for longerduring polymerization and hence are more prone to distortion due toshear or collision. The internal morphology of these beads also appearedto be more open and porous than that of polymer PF2. This mightcontribute to the better HPLC performance of the latter. The beads ofFIG. 2(c) show PF13 beads and FIG. 2(e) PF15 beads.

High Pressure Liquid Chromatography

To confirm that the polymer beads made according to the presentinvention are molecularly imprinted, and that the quality of therecognition sites is at least as good as that obtained by traditionalbulk polymerization methods, a range of polymers imprinted using Boc-Phewere evaluated by HPLC. This system was chosen since a great deal ofinformation is available on the performance of traditional crushed bulkpolymers imprinted with Boc-Phe.

Beads were suspended in a chloroform-acetone (17:3) mixture bysonication and slurry packed into 10 cm by 0.46 cm or 25 cm by 0.46 cmstainless steel columns at 300 bar using an air driven fluid pump andacetone as solvent. The columns were washed with 250 ml methanol: aceticacid (9:1) and then equilibrated with chloroform containing 0.1% or0.25% acetic acid. 10 g Boc D- or L- Phe or 20 μg racemate in 20 μlsolvent was injected and chromatograms recorded at 254 nm at a flow rateof 0.5 ml/min. Some separations were also run at higher flow rates andwith larger amounts of compound loaded. Chromatographic parameters werecalculated using standard theory.

The results for HPLC evaluation of six of the polymers are summarized inTable 2.

                  TABLE 2                                                         ______________________________________                                        ID†                                                                          Ratio‡                                                                      % x-link K'D  K'L   Alpha Rs   f/g                             ______________________________________                                                      CHCl.sub.3 + 0.1% Acetic Acid                                   PF2   1:4      80       0.69 1.44  2.09  0.59 0.51                            PF8   1:8      71       0.77 1.42  1.88  0.43 0.37                            PF9   1:12     62.5     1.12 1.88  1.68  0.83 0.73                            PF10  1:16     56       1.44 2.81  1.81  0.49 0.5                             PF11  1:12     75       0.71 1.27  1.8   0.87 0.63                            PF11* 1:12     75       0.79 1.43  1.82  1.23 0.84                            PF12  1:4      80       0.7  0.7   0     0    0                               ______________________________________                                                      CHCl.sub.3 + 0.25% Acetic Acid                                  PF2   1:4      80       0.43 0.78  1.81  0.26 0.23                            PF8   1:8      71       0.54 0.97  1.79  0.31 0.28                            PF9   1:12     62.5     0.76 1     1.7   0.69 0.66                            PF10  1:16     56       0.5  1.89  1.89  0.48 0.44                            PF11  1:12     75       0.63 0.71  1.78  0.48 0.39                            PF11* 1:12     75       0.84 1.11  1.91  1.08 0.88                            PF12  1:4      80       0    0.54  0     0    0                               ______________________________________                                         †The identification scheme (ID) for the polymers is consistent wit     the notation developed previously at Table 1.                                 ‡The ratio shown is that of the print molecule to the              methacrylic acid (MAA) monomer.                                          

As the ratio of MAA to print molecule increased the retention times andhence the capacity factors increased due to greater non-specificinteraction. The α-values, however, stayed almost constant at about 1.8.This is very similar to values obtained under similar conditions forground and sieved bulk Boc-Phe polymers (range 1.77 to 2.17). Theoptimum resolution was found at an MAA:Boc-Phe ratio of 12:1, the valueof 0.83 being good for a 10 cm column. Using a longer column (25 cm) ofpolymer PF11 resulted in near baseline resolution of the enantiomers ashas previously been reported for bulk polymers (chromatogram not shown).Polymer PF9, which was only 62.5% cross-linked, performed better thanpolymer PF11, which had the same print molecule:MAA ratio but was 75%cross-linked. It has previously been shown that separations improve asthe degree of cross-linking increases within this range, but this wasnot observed in these experiments. Polymer PF11 was made using lessprint molecule (see Table 1) since it is not possible too vary theseparameters independently, and it is thus not clear whether the improvedresolution was due to the larger number of binding sites or to changesin polymer morphology as a result of the lower cross-linking. Suchobservations indicate that significant improvements in separation can beachieved by careful optimization of the many compositional andoperational variables. The simplicity and speed of the beadpolymerization method makes extensive optimization possible.

It has previously been suggested that polymers based on thetrifunctional cross-linker TRIM had much better resolution and loadcapacity than EDMA-based polymers for a range of di and tripeptides. Inorder to further evaluate the suspension polymerization method accordingto the present invention, imprints of Boc-L-Phe were made in aTRIM-based polymer. Beads produced using 100 mg of PFPS (PF15) had anaverage diameter of 5.7 cm, and those using 25 mg PFPS (PF16) a diameterof 18.8 μm, very similar to what would have been expected for EDMA-basedpolymers with the same amount of stabilizing polymer. Thus, in terms ofbead-size prediction, TRIM and EDMA seem to behave very similarly. AnSEM picture of beads of PF15 is shown in FIG. 2e. These beads weretested by HPLC and gave excellent resolution and high load capacities,as was noted for the ground and sieved block polymers. The packed columnhad very low back pressure, and high resolution could be achieved, evenat quite high flow rates.

FIGS. 3a-3e shows a series of chromatograms for flow rates between 0.5and 5 mlmin⁻¹, i.e., 0.5 ml/min, 1 ml/min, 2 ml/min, 3 ml/min and 5ml/min. Little difference was observed between 0.5 and 2 mlmin⁻¹,suggesting that diffusion rates are rapid for these small beads. Theback pressure was very low and resolution excellent (fg=0.89, 0.89 and0.85 at 0.5, 1 and 2 mlmin-1 respectively). Reasonable resolution(f/g=0.61) was still achieved at 5 mlmin-1 (back pressure 1300 psi).Ground and sieved random <25 μm particles do not usually perform well atflow rates above 1 mlmin-1. Working with crushed bulk polymers in the 5μm size range is difficult. Extensive defining is required to avoid highback pressures in HPLC columns, and sieves below 10 μm are unobtainable,making it necessary to use alternative size fractionation techniques.This result indicates that the beaded imprinted polymers should havesignificant advantages over "traditional" ground and sieved blockpolymers, both in the case of preparation and in the performance of theresulting columns.

It is also contemplated that because the stabilizer of the presentinvention is essentially chemically inert, it may be used as adispersant for making beaded polymers containing water-sensitive monomerunits, e.g., acid chlorides or anhydrides.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope thereof as described in the specification andas defined in the appended claims.

We claim:
 1. A suspension polymerization stabilizer comprising acopolymer containing monomer units A defined as C_(n) F_(2n+1) SO₂ N(C₂H₅)C₂ H₄ --O--CO--CH═CH₂ and comonomer units B defined as CH₃ O(CH₂ CH₂O)_(m) C₂ H₄ --O--CO--CH═CH₂ ; where n is between 1 and 20 and m isbetween 10 and 70 and about
 500. 2. The stabilizer according to claim 1,wherein n is about 7.5 and m is about
 43. 3. A polymer defined by theformula ##STR5## wherein X is C_(n) F_(2n+1), C_(n) F_(2n+1) (CH₂)_(r)O--, C_(n) F_(2n+1) O-- or C_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₂ --O;Y is(Z)_(t) CH₃ O(CH₂ CH₂ O)_(m) C₂ H₂ --O or (Z)_(t) CH₃ O(CH₂ CH₂ O)_(m)-- Z is a print molecule; n is between 1 and 20; m can be zero to about500; p is at least 1; q can be zero or any positive number; r is 1-20,preferably 1 or 2; s is zero or 1; and t is zero or
 1. 4. A polymerdefined by the formula ##STR6## wherein X is C_(n) F_(2n+1) SO₂ N(C₂H₅)C₂ H₂ --O;Y is CH₃ O(CH₂ CH₂ O)_(m) C₂ H₂ --O; n is between 1 and 20;m is between 0 and 500; p is at least 1; and q zero or any positiveinteger.
 5. The stabilizer according to claim 1, wherein n is about 7.5and m is about
 43. 6. A stable emulsion comprising an emulsion ofimprinting molecules in liquid perfluorocarbons containingpolyoxyethylene ester groups.
 7. The emulsion according to claim 6,wherein said liquid fluorocarbon is a copolymer.
 8. The emulsionaccording to claim 7, wherein said copolymer is formed from thepolymerization of monomer A and comonomer B, wherein monomer A isdefined as C₂ F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ --O--CO--CH═CH₂, and comonomerB is defined as CH₃ O(CH₂ CH₂ O)_(m) C₂ H₄ --O--CO--CH═CH₂, where n isbetween 1 and 20, and m zero to about
 500. 9. An emulsion comprisingimprinting molecules and a polymer defined by the formula ##STR7##wherein X is C_(n) F_(2n+1), C_(n) F_(2n+1) (CH₂)_(r) O--, C_(n)F_(2n+1) O-- or C_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ --O;Y is (Z)_(t) CH₃O(CH₂ CH₂ O)_(m) C₂ H₄ --O or (Z)_(t) CH₃ O(CH₂ CH₂ O)_(m) -- Z is aprint molecule; n is between 1 and 20; m can be zero to about 500; p isat least 1; q can be zero or any positive number; r is 1-20, preferably1 or 2; s is zero or 1; and t is zero or
 1. 10. An emulsion comprisingimprinting molecules and a polymer defined by the formula ##STR8##wherein X is C_(n) F_(2n+1) SO₂ N(C₂ H₅)C₂ H₄ --O;Y is CH₃ O(CH₂ CH₂O)_(m) C₂ H₄ --O; n is between 1 and 20; m is zero to about 500; p is atleast 1; and q is zero or any positive integer.